Establishment of a Mouse Severe Acute Pancreatitis Model using Retrograde Injection of Sodium Taurocholate into the Biliopancreatic Duct

Summary

A mouse model of severe acute pancreatitis is described herein. The procedure presented here is very rapid, simple, and accessible, thereby potentially allowing the study of the molecular mechanisms and different therapeutic interventions in acute pancreatitis in a convenient way.

Abstract

The prevalence of acute pancreatitis (AP), especially severe acute pancreatitis (SAP), is increasing in younger age groups annually. However, there is a lack of effective treatments in the current clinical practice. With the easy accessibility of transgenic and knockout strains and their small size, which allows minimal doses of drugs required for in vivo evaluation, a well-established experimental model in mice is preferred for AP research. Moreover, SAP induced through sodium taurocholate (TC) is currently one of the most widely used and best characterized models. This model has been investigated for novel therapies and possible molecular events during the process of AP. Here, we present the generation of an AP mouse model using sodium taurocholate and a simple homemade microsyringe. Moreover, we also provide the methodology for the subsequent histology and serological testing.

Introduction

Acute pancreatitis (AP) is an acute inflammation of the pancreas characterized by obstruction of the main pancreatic duct with subsequent ductal distension and pancreas autodigestion by its abnormally activated enzymes. Its clinical manifestations include local or systemic inflammation, abdominal pain, and elevation of serum amylase^1,2. According to the severity classification³, AP can present in mild, moderate, and severe forms, and among them, severe acute pancreatitis (SAP) is the most concerning condition due to its high mortality rate of more than 30%⁴. In the United States, AP is one of the most common reasons for hospitalization, affecting over 200,000 patients⁵. Moreover, AP, especially SAP, is increasing annually and affecting younger age groups⁶. However, there is a lack of effective treatment options in current clinical practice^6,7. Therefore, it is necessary to explore the molecular mechanisms involved in AP, thereby facilitating treatment improvement.

Well-established experimental animal models are required for studying the mechanisms involved in AP and evaluating the effectiveness of different treatment modalities. With the easy accessibility of transgenic and knockout strains and their small size, which minimizes the doses of drugs required for in vivo evaluation, mice are preferred for AP research. Therefore, several models of AP have been developed in mice^8,9.

Working from a mild pancreatitis rat model induced through the intravenous administration of caerulein¹⁰, Niederau et al. developed a SAP mouse model presented with acinar cell necrosis induced using the same drug and injection route¹¹. Although this model possesses several advantages, including noninvasiveness, rapid induction, wide reproducibility, and applicability, the major disadvantage is that only a mild form of AP is developed in most cases, thereby limiting its clinical relevance. Alcohol is considered one of the major etiologic factors of AP; however, Foitzik et al. reported that it causes pancreatic injury only when combined with other factors, such as exocrine hyperstimulation¹². Moreover, although alcohol-induced AP models developed via different administration routes, and drug doses have been reported^13,14,15, their major disadvantage is the difficulty in reproducing them. Intraperitoneal administration of L-arginine can also induce AP in mice¹⁶; however, its low clinical relevance hinders its application. Taurocholate, a bile salt, was first proposed by Creutzfeld et al. in 1965 for inducing a condition resembling human AP via pancreatic duct infusion¹⁷. Although controversies exist regarding its clinical relevance in pathophysiology^18,19, taurocholate-induced pancreatitis remains an indispensable model for SAP.

As this model is simple to realize and is also effective in mice, it is now one of the most used AP models for small animal in vivo studies. Perides et al. employed sodium taurocholate (TC) to induce SAP in mice²⁰, providing insights to understand its pathology. Combined with genetic modification techniques, this model has allowed us to confirm several specific genes involved in AP. For example, Bicozo et al. showed that a knockout of the CD38 gene protected against a model of TC-infusion pancreatitis and attributed the mechanisms to alterations in intracellular Ca²⁺ signaling²¹. Fanczal et al. investigated the physiological implication of TRPM2 expression in the plasma membrane of mouse pancreatic acinar and ductal cells, and demonstrated reduced severity of TC-induced SAP in TRPM2 knockout mice²². Furthermore, this model also provides a simple and effective way to test many novel drugs in vivo. For instance, this method enabled validation of the therapeutic effects of caffeine²³, dehydrocholic acid²⁴, and various antioxidants and anticoagulants^25,26. This evidence demonstrates the versatility of the TC-induced SAP model. Although Wittel et al. described a similar mouse model²⁷, a lack of details on the implementation procedures could result in an inability to reproduce the findings. In this article, we focus on methods using a simple homemade microsyringe and study TC-induced SAP, thereby providing possible guidance not only for further study of the pathogenesis and treatment of AP, but also for a perfectly adaptable experimental method for many other substances.

Protocol

All experiments involving animals were approved by the Animal Ethics Committee of Soochow University. All surgical procedures were performed under full anesthesia. Analgesics were not used to avoid interference with the natural course of the disease according to previous literatures^28,29. Approval for the lack of analgesia was also granted by the Animal Ethics Committee of Soochow University.

1. Preparation

1. Fast a C57BL/6 wild-type mouse 8-12 h before the surgery.
2. Prepare 5% (w/v) TC solution (93 mM) diluted into 0.9% sodium chloride. Filter the solution through a 0.22 µm filter.
3. Autoclave the surgical instruments and materials, including forceps, scissors, blade holder, needle holder, hemostatic clip, vascular clamps, and sterile drapes, using a pressure sterilizer at a temperature of 121 °C for 30 min.
4. Prepare a homemade microsyringe. To do this, manually install a disposable insulin tip and a prolonged plastic pipette tip onto a 25 µL flat tip Microliter syringe. Sterilize through irradiation.

2. Anesthesia and preoperative preparation

1. Weigh 6- to 8-week-old male C57BL/6 wild-type mice (approximately 21-23 g). Induce general anesthesia with an intraperitoneal injection of 1% pentobarbital sodium solution at a dose of 50 mg/kg.
2. Fix the mouse in the supine position on anirradiatedfoam flat plate with the tape.
3. Prepare the operation region of each animal by removing its fur with a depilation cream from the xiphoid process level down to the connecting line of the bilateral anterior superior iliac spine, with the left and right sides parallel to the front axillary line.
4. Prepare a sterile surgical field with particular focus on the surgical area, consisting of the area prepared on the animal and the front of the surgeon.
   1. Disinfect the benchtop.
   2. Clean the incision site with multiple applications of surgical scrub solution. Wipe the skin two to three times with 0.5% iodophor and allow it dry for 1-2 min each time.
   3. Cover the mouse with sterile drapes.

3. Surgical procedure

NOTE: Figure 1 depicts the anatomy and morphology of the mouse pancreas before and after retrograde injection of TC into the biliopancreatic duct by microinjection.

1. Make an approximate 2 to 3 cm long median abdominal incision.
2. Locate the duodenum from left to right along the stomach. Gently pull the duodenum out and to the left side using surgical forceps and a disinfectant-soaked cotton swab to expose the pancreas, the biliopancreatic duct, and the duodenal papilla. Take care not to injure the intestinal wall or related vessels.
3. Use 8-0 sutures to pass through the connection between the duodenum and pancreas around the papilla, and pull them to the left side of the mouse body to fix the duodenum.
4. Gently pull the skin and the lower edge of the liver aside. Clamp the common hepatic duct, making sure not to clamp the portal vein or pancreas. Clamp the proximal biliopancreatic duct.
5. Pierce the microinjector into the distal biliopancreatic duct retrogradely. Fix the needle tip near the duodenal papilla with a clamp. Infuse 10 µL of 5% TC into the pancreatic duct at a rate of 5 µL/min.
6. Keep the biliopancreatic duct closed for 5 min to achieve a stable pressure. Then, pull out the microinjector and remove the clamps.
7. Restore the duodenum to the original anatomical position and check the bleeding.
8. Close the wound with 6-0 sutures and disinfect with 0.5% iodophor.

4. Recovery and postoperative management

1. Recover the animals from anesthesia on a 37 °C thermal pad. Do not send the operated animals to the temporal postoperative room until all the animals have woken up.
2. Administer 0.8 mL of 0.9% saline subcutaneously and slowly for fluid supplementation.
3. Monitor the animals postoperatively for their general condition and signs of infections or illness.

5. Serological testing and histology evaluation

1. At 24 h post-operation, anesthetize the mice with pentobarbital sodium using the same dose and injection route.
2. Collect all the blood from the abdominal aorta (about 0.8 mL per mouse) and spin at 1,500 x g for 15 min to isolate the serum for further testing.
3. Collect the pancreas tissues.
4. Open the thoracic cavity to collect the lung tissues.
5. Measure the biochemistry function indexes from the serum, including amylase, lipase, liver function (alanine transaminase (ALT) and aspartate transaminase (AST)), and kidney function (urea and creatine), using commercial kits according to the manufacturers' instructions.
6. Homogenize the lung and pancreas tissues and measure the myeloperoxidase (MPO) level using a commercial kit according to the manufacturer's instructions.
7. Open the thoracic cavity and perfuse with 20 mL of phosphate-buffered saline (PBS) twice, then collect the pancreas. Immerse into 4% paraformaldehyde solution for 24 h. Dehydrate in a series of alcohol concentrations and embed in paraffin.
8. Use a microtome to prepare 5 µm thick pancreas tissue sections.
9. Perform H&E staining for histological evaluation of the pancreas.
10. Evaluate the pathological scores according to the criteria proposed by Schmidt et al.³⁰.

Results

By carefully following the instructions above, we obtained a mean surgery duration of approximately 40 min. The mice were slightly inactive and had lost approximately 0.5-1.75 g, 0.85-1.85 g and 0.5-4.73 g of weight at 24 h, 48 h and 72 h post-operation, respectively (Figure 2).

From the time of surgery completion to 24 h post-operation, as the disease developed, the mice became inactive and showed slow responses and actions.

The surviv...

Discussion

The TC-induced SAP model is an excellent research tool. As shown in this study, this model is very easily realized in general labs without employing specific devices. When used in combination with histology and biochemical analysis, it provides a cost- (inexpensive reagents) and time-saving (24 h time window) approach for inducing and evaluating AP. Adjusting the concentration of TC also offers the possibility of producing mild and moderate AP. Perides et al. also employed TC to induce SAP in mice²⁰

Materials

Name	Company	Catalog Number	Comments
0.5% iodophor	Shanghai Likang Disinfectant	310102	4 mL/mouse
0.9% sodium chloride	Sinopharm Group Co., Ltd.	10019318	0.8 mL/mouse
1% Pentobarbital sodium	Sigma	P3761	0.2 -0.25 mL/mouse
25 μL flat tip Microliter syringe	Gaoge, Shanghai	A124019
4% Paraformaldehyde	Beyotime, Nantong, China	P0099-500ml
5% sodium taurocholate (TC)	Aladdin	S100834-5g	10 μL/SAP mouse
6-0 Sterile nylon microsuture with threaded needle (1/2 circle)	Cheng-He	20093
75% alcohol	Sinopharm Group Co., Ltd.	10009218	4 mL/mouse
8-0 Sterile nylon microsuture with threaded needle (3/8 circle)	Cheng-He	19064
ALT Activity Assay Kit	EPNK, Anhui, China	ALT0012
Amylase Assay Kit	EPNK, Anhui, China	AMY0012
Angled small bulldog clamp with 12 mm jaw (3 cm)	Cheng-He	HC-X022
aspen shavings or shreds for mouse bedding	Beijing Vital River Laboratory Animal Technology	VR03015
AST Activity Assay Kit	EPNK, Anhui, China	AST0012
Blood Urea Nitrogen (BUN) Assay Kit	EPNK, Anhui, China	BUN0011
C57BL/6 mouse	Beijing Vital River Laboratory Animal Technology	213
Creatine Assay Kit	EPNK, Anhui, China	CRE0012
Feature microtome blade	Beyotime, Nantong, China	E0994
Hemostatic Forceps (9.5 cm, Curved)	JZ, Shanghai Medical Instruments Co. Ltd.	JC3901
Lipase Assay Kit	Jiancheng, Nanjing, China	A054-2-1
Microtome	Leica biosystem, Germany	RM2245
Mindray biochemistry analyzer	Mindray, Shenzhen, China	BS-420
MPO Assay Kit	Jiancheng, Nanjing, China	A044-1-1
Normal mouse chow	Trophic, Nantong, China	LAD 1000
Phosphate buffered saline	Beyotime, Nantong, China	C0221A
Straight micro-bulldog clamp with 5 mm jaw (1.5 cm)	JZ, Shanghai Medical Instruments Co. Ltd.	W40130
Straight or curved forceps (11.0 cm)	Cheng-He	HC-X091A or HC-X090A
Straight Scissors (10.0 cm)	Cheng-He, Ningbo, China	HC-J039102
Thermo Scientific Centrifuge	Thermo Scientific, USA	Multifuge X1R